The Lys-PCLA bioconjugates are prepared using thiol-ene reaction between thiolated lysozyme (Lys-SH) and acrylated PCLA (PCLA-Ac). The free-flowing Lys-PCLA bioconjugate sols at low temperature transformed to immovable gel at the physiological condition and exhibited stability upon dilution with buffers. According to the in vitro toxicity test, the Lys-PCLA bioconjugate and PCLA copolymer were non-toxic to RAW 263.7 cells at higher concentrations (1000 µg/mL). In addition, subcutaneous administration of Lys-PCLA bioconjugate sols formed stable hydrogel depot instantly, which suggested the in situ gel forming ability of the bioconjugate. Moreover, the Lys-PCLA bioconjugate hydrogel depot formed at the interface between subcutaneous tissue and dermis layers allowed the active migration and recruitment of DCs. As suggested by these results, the in-situ forming injectable Lys-PCLA bioconjugate hydrogel depot may serve as an implantable immune niche for the recruitment and modification of DCs.Despite the potential of hydrogel-based localized cancer therapies, their efficacy can be limited by cancer recurrence. Therefore, it is of great significance to develop a hydrogel system that can provoke robust and durable immune response in the human body. This study has developed an injectable protein-polymer-based porous hydrogel network composed of lysozyme and poly(ε-caprolactone-co-lactide)- Hydrogels, as water-swollen three-dimensional amphiphilic or hydrophilic polymer networks, are used for the encapsulation and controlled release of chemotherapeutic drugs, therapeutic proteins as well as peptides, nucleic acid and cells ,2,3,4. OIt has been found that the implantation of hydrogels into the body is a suitable strategy to cure various kinds of acute and chronic diseases , but the4+ and CD8+ cells, respectively, they are considered as the key regulators in initiating the immune response [In recent years, injectable hydrogels and scaffolds have been explored in a wide range of immunological applications ,33,34. Gresponse . Hence, b-poly(ethylene glycol)-b-poly(ε-caprolactone-co-lactide (PCLA-PEG-PCLA) (hereafter referred as PCLA) conjugated lysozyme (Lys-PCLA) bioconjugate for the active recruitment and modulation of dendritic cells (DCs) -ls (DCs) . Lysozymbacteria . In thisn = 1500 g/mol), DL-lactide (LA), ε-caprolactone (CL), triethyl amine (TEA), acryloyl chloride, and stannous octoate (Sn(Oct)2) were supplied by Sigma-Aldrich .Lys from chicken egg white, tris(2-carboxyethyl)phosphine hydrochloride salt (TCEP), PEGs (MThe Michael reaction between acrylated PCLA (PCLA-Ac) and thiolated Lys (Lys-SH) was used to prepare Lys-PCLA bioconjugate. Meanwhile, the synthetic route to obtain Lys-PCLA bioconjugate is shown in Preparation of PCLA copolymer: The PCLA copolymer was synthesized by following the previously reported procedure [2  were placed and stirred for 3 h at 110 °C. Afterwards, the temperature was decreased to 60 °C. Subsequently, CL  and LA  were added and allowed to polymerize at 130 °C for 18 h. One day after the reaction, chloroform was added to reduce the viscosity of the reaction mixture and the product was precipitated using the n-hexane and diethyl ether mixture . Yield: 79%.rocedure . BrieflyPreparation of PCLA-Ac: Hydroxyl chain-end of the PCLA copolymer reacted with acryloyl to introduce acrylate groups. Briefly speaking, PCLA (1 mmol) was dissolved in toluene in the presence of TEA (3 mmol). Apart from that, the solution was stirred at 0 °C. Acryloyl chloride (3 mmol) in toluene was added dropwise and then stirred for 48 h. Furthermore, the PCLA-Ac copolymer was obtained by precipitating the reaction mixture in diethyl ether. Yield: 89%.Preparation of Lys-SH: Lys was dissolved in 2 M urea containing PBS buffer solution at a concentration 5 mg/mL under the nitrogen environment. Afterwards, TCEP (14 eq.) was added adjusted to 8.0 in terms of pH and stirred at 0 °C for 1 h. The Lys-SH was obtained by passing it through a PD-10 desalting column and then the product was stored at 0 °C. Yield: 92%.Preparation of Lys-PCLA bioconjugates: The Lys-PCLA bioconjugate was prepared using the Michael-addition reaction between Lys-SH (1 mmol) and PCLA-Ac (3 mmol). In brief, PCLA-Ac was dispersed in PBS at 0 °C (10 wt.% concentration). Subsequently, Lys-SH was added and stirred at 0 °C. After two days, the reaction mixture was moved to the cellulose membrane (MWCO: 3500 kDa) and extensively dialyzed against deionized water for three days. In order to avoid the Lys-PCLA bioconjugate aggregation, the dialysis was performed at 0 °C. Finally, Lys-PCLA bioconjugate was acquired by lyophilization. Yield: 76%.1H NMR analysis: The chemical structure and number-average molecular weight (Mn) of copolymers were confirmed using 1H NMR spectra . In order to record 1H NMR, the PLCA copolymer or PLCA-Ac copolymer (1 wt.%) had been dissolved in CDCl3.FT-IR: FT-IR spectra of the copolymer and bioconjugate were obtained using an FT-IR instrument  within the scanning range of 400–4000 cm−1.Scanning electron microscope (SEM): The surface morphology of hydrogels was observed using SEM . After hydrogel samples were placed on SEM stubs, samples were sputter-coated with platinum to make them conductive.Gel permeation chromatography (GPC): The number-average molecular weight (Mn) and polydispersity index (PDI) of PCLA copolymers were measured using Waters GPC  using polystyrene standards.The tube inversion method was adopted to investigate the flow (sol) and non-flow (gel) behavior of PCLA copolymers and Lys-PCLA bioconjugates . In ordeIn order to examine the mechanical strength of hydrogels prepared from PCLA copolymers and Lys-PCLA bioconjugates, the mechanical properties including viscosity, storage modulus (G′), and loss modulus (G″) were measured using a Bohlin Rotational Rheometer equipped with a parallel steel plate (40 mm) and a Peltier system to control the temperature. For the rheological measurement, 22.5 wt.% of either PCLA copolymer or Lys-PCLA conjugate was prepared, as described in the previous section, and the samples were placed between the parallel plates with a plate gap of 0.25 mm.gel) was determined by raising the temperature from 10 °C to 60 °C at a constant frequency (1 Hz) and shear stress (0.4 pa) with a heating rate of 2 °C/min. The G′ and G″ were measured as a function of temperature and the Tgel was determined from the intersection point of the curves. The complex viscosity was also measured without changing the measurement parameters.The gelation temperature (TIn order to determine the linear viscoelastic region (LVER) and observe the point at which the hydrogel structure begin to deteriorate, the strain sweep experiments were performed from 1% to 100% at a constant frequency (1 Hz) and temperature (37 °C). The frequency sweep test was also conducted from 0 to 50 Hz within the LVER.w/v)). Beyond that, the cell flasks were incubated at 37 °C in a humidified 5% CO2-95% air atmosphere.The RAW 263.7 cells were purchased from Korean Cell Line Bank (KCLB) and cultured in a DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin  was added and incubated for 3 h at 37 °C. At the same time, the purple crystals were dissolved in DMSO and the viability was examined with a Microplate reader by measuring the absorbance at 490 nm. The RAW 263.7 cells cultured with only fresh culture medium were taken as the control.In order to determine the biocompatibility of copolymers and bioconjugates, the RAW 263.7 cells, at a density of 10In order to investigate the in situ gelation properties of hydrogels, six-week-old SD rats with an average weight of 225 g were employed. In vivo gelation was confirmed by subcutaneously administering 300 µL (22.5 wt.%) of PCLA copolymers or Lys-PCLA conjugate solutions into the back of SD rats. Shortly before use, the hydrogel precursors were transferred to a hypodermic syringe equipped with a 26G needle and kept in an ice bath to avoid early gelation. In addition, the injection site of the SD rats was shaved and sterilized using 70% ethanol. Ten minutes after the injection, the SD rats were sacrificed. The injection site was cut-open to recover the hydrogels recovered and photographed. Afterwards, the recovered hydrogels were frozen and freeze-dried. The freeze-dried products were cross-sectioned and coated with platinum using a spin coater and then the porous structure was visualized using SEM.In order to analyze the recruitment of the cell in the hydrogel network, either Lys-PCLA solution or PCLA solution  was subcutaneously injected to BALB/c mice and allowed to recruit cells for 3 days. Thereafter, the hydrogel nodules were collected and the cells were counted by digesting hydrogels with collagenase type II solution. Furthermore, the hydrogels debris was filtered by a 20 µm cell trainer and subsequently washed twice with a buffer. Afterwards, FcR blocking reagent was adopted to count and stain the cells. Subsequently, the cells were stained using mouse monoclonal antibodies against CD11c and CD11b in a FACS buffer followed by flow cytometry analysis.All the animal experiments were followed according to the Sungkyunkwan University standards protocols and the Institutional Committees of Sungkyunkwan University approved all the animal experiments performed in this study.t-test.The statistical significance between the groups is analyzed using a two-tailed Student N-isopropylacryamide) and polyphosphazene, have often been studied because of their sharp transition properties [The design of injectable hydrogels that could recruit and modulate immune cell response has been a hot spot of research . Among toperties . In our operties ,53,54. H1H NMR spectra. 1H NMR spectra show the PCLA copolymer featuring characteristics peaks of PEG, CL and LA components (2-CH2-O-) appeared at 3.63 ppm, whereas the methylene proton next to the carbonyl appeared at 2.29 ppm. At the same time, the methine proton that originated in lactide units appeared at 5.13 ppm. The molar composition and Mn of the PCLA copolymer was calculated by comparing the characteristic peaks of PEG at 3.63 ppm with CL and LA characteristic peaks at 2.29 ppm and 5.13 ppm, respectively. Then, PCLA-Ac was prepared by acrylation of chain-end hydroxyl functional groups in the presence of triethylamine base. 1H NMR shows the appearance of new peaks at 5.76 to 6.66 ppm corresponding to the characteristic peaks of acrylate, which indicates successful acrylation of PCLA copolymers  . The preco-lactide) block is hydrophobic, while the PEG block is hydrophilic in the copolymer that induces micelle formation. It is noteworthy that PEG also exhibited mild hydrophobic interactions with increasing temperature driven by the loss of orientational entropy in which the ordered interaction between PEG and water molecules was disordered. This not only resulted in entropy change (DS > 0), but also led to the hydrophobic aggregation. Thus, with the increase of the temperature, the hydrophobic aggregation was strengthened and under certain conditions such as concentrations, hydrophobic and hydrophilic balance, and medium PCLA and other poly(ester)-based copolymer underwent sol-to-gel phase transition  of PCLA and Lys-PCLA hydrogels, the complex viscosity and modulus (G′ and G″) of hydrogels at different temperatures have been examined. As expected, at 22.5 wt.%, both PCLA and Lys-PCLA bioconjugates changed in viscosity with the increase of temperature  and loss modulus (G″) crossover occurred. gel of Lys-PCLA and PCLA hydrogels was found to be 20.50 °C and 24.10 °C, respectively. The slightly low temperature gelation from Lys-PCLA hydrogels was due to non-covalent cross-linking between Lys and PCLA copolymers, which accelerated the gelation process. The mechanical properties of hydrogels are the key characteristics in tissue engineering and controlled delivery applications. In particular, the G′ of both Lys-PCLA and PCLA hydrogels was higher than the G″ when the temperature was raised over 20 °C, which implies that elastic modulus was higher at this temperature. The G′ value of Lys-PCLA at 37 °C was close to 10,000 Pa which showed the good stiffness of the gel similar to other reported hydrogel formulations [The Tulations .Furthermore, oscillatory strain-sweep and frequency-sweep measurements were carried out as a function of shear strain and frequency, so as to determine the LVER and robustness of the hydrogels. The oscillatory strain-sweep test was performed on a preformed hydrogel at a constant frequency of 1 Hz at 37 °C from 1% to 100% strain amplitude. As shown in Hydrogels are generally considered as soft materials with viscoelastic behavior. The viscosity of both PCLA and Lys-PCLA hydrogels is low and shows no significant changes in viscosity between 0 °C and 20 °C, which indicates the Newtonian flowing of hydrogels. Interestingly, when the temperature was raised over 20 °C, the viscosity started increasing gradually and reached a maximum close to the physiological temperature. This suggested the non-Newtonian flowing of the hydrogels. Furthermore, the oscillatory temperature-sweep, strain-sweep and frequency-sweep tests are often employed to investigate the viscoelastic property of a hydrogel. In Non-toxicity of a hydrogels is an essential factor for biomaterials in in vivo applications. To examine biocompatibility, different concentrations of PCLA copolymers or Lys-PCLA bioconjugates were exposed to RAW263.7 macrophages and their viability was evaluated with an MTT assay. In general, hydrogels exhibit numerous interesting properties such as non-toxicity, self-healing, biocompatibility, and biodegradability. Among them, biocompatibility is an important prerequisite for any hydrogels injected into the living body. Therefore, these characteristics are frequently demanded and the hydrogels with such properties are given attention in medical applications. When the hydrogels are applied into the body, the biocompatibility possible toxic effect of degraded byproducts need to be considered. In situ forming injectable hydrogels developed in this study are based on biodegradable poly(ester), such as PCLA. The PCLA-based hydrogels developed in this study show little or no toxicity at high concentrations (1000 µg/mL). This result was in agreement with our previous report based on PCLA hydrogels . It shouWith the promising phase transition and good biocompatibility in vitro, the in situ gel formation was examined in warm-blooded animals. The in situ gel formation was investigated by locally injecting the Lys-PCLA bioconjugate sols or PCLA copolymer sols  into the subcutaneous layer of SD rats using a 26 G hypodermic needle. The rats were sacrificed 10 min after injection and the injection site was cut open to investigate the gelation. As observed in The subcutaneously formed hydrogels were recovered and subjected to SEM imaging to confirm surface morphology, porosity, and diffusivity of the hydrogels. The SEM images of both Lys-PCLA and PCLA hydrogels are shown in As shown in Numerous locally injectable hydrogels have been developed for tumor immunotherapy. In particular, gelatin-based hydrogels are widely used in various biomedical applications because their properties often mimic the natural properties of the extracellular matrix. Various gelatin-based cell adhesive and degradable hydrogels have been developed and were shown to have good cell attachment, proliferation, and survival properties. Koshy et al. developed injectable porous gelatin cryogels and they were found to be minor host responsive following subcutaneous injection . ControlThis study developed an in situ forming injectable Lys-PCLA bioconjugate hydrogel for the in situ recruitment of DCs. The presence of natural Lys protein in the hydrogel network endows a good porous mesh-like network. The Lys-PCLA hydrogel is non-toxic to macrophages even at 1000 µg/mL concentrations. Lys-PCLA hydrogels exhibited good sol-to-gel phase transition and good mechanical properties. The hydrogel endows a good in situ gel forming ability into the back of rats. Subcutaneous administration of Lys-PCLA bioconjugate sols into the back of mice spontaneously forms a hydrogel network. Owing to the porous properties, various immune cells including DCs and macrophages migrated to the porous network. The cells are modulated by the introduction of appropriate antigens, which can control the antitumor immunity. As suggested by the results, Lys-PCLA conjugate is a powerful alternative to therapies that require host cell modulation or the in situ reprogramming of host cells.


Conventional wisdom suggests mid-task rest as a potential approach to relieve the time-on-task (TOT) effect while accumulating evidence indicated that acute exercise might also effectively restore mental fatigue. However, few studies have explored the neural mechanism underlying these different break types, and the results were scattered. This study provided one of the first looks at how different types of fatigue-recovery break exerted influence on the cognitive processes by evaluating the corresponding behavioral improvement and neural response  in a sustained attention task. Specifically, 19 participants performed three sessions of psychomotor vigilance tasks (PVT), with one session including a continuous 30-min PVT while the other two sessions additionally inserted a 15-min mid-task cycling and rest break, respectively. For behavioral performance, both types of break could restore objective vigilance transiently, while subjective feeling was only maintained after mid-task rest. Moreover, divergent patterns of EEG change were observed during post-break improvement. In detail, relative theta decreased and delta increased immediately after mid-task exercise, while decreased delta was found near the end of the rest-inserted task. Meanwhile, theta and delta could serve as neurological indicators to predict the reaction time change for exercise and rest intervention, respectively. In sum, our findings provided novel evidence to demonstrate divergent neural patterns following the mid-task exercise and rest intervention to counter TOT effects, which might lead to new insights into the nascent field of neuroergonomics for mental fatigue restoration. In contemporary society, sustaining steady attention for a long period is a challenging yet necessary cognitive process on numerous occasions  on mental fatigue recovery and indicated that longer breaks were associated with better restoration of behavioral performance  paradigm was used in this work. Specifically, a within-subject three-session design was adopted, with one session consisting of a continuous 30-min PVT while the other two sessions additionally included a 15-min mid-task rest break and a 15-min cycling break, respectively. Unlike our previous research that focused on network analysis and revealed non-significant effects  were included for the following analysis. All participants had normal or corrected-to-normal vision. None of them reported cardiovascular, cognitive disorder, color blindness, or any acute illness that might influence the cognitive task performance. Each participant was informed of the experimental protocol and potential risks through a telephone interview. They were required to get a full night of sleep  for two continuous nights and were instructed to abstain from caffeine and alcohol and avoid vigorous physical activity for 7 h before the experiment. Those participants who failed to meet the requirements were either ruled out from the experiment or rescheduled. Upon arriving at the lab, written informed consent and Physical Activity Readiness Scale (PARS) were provided to exclude any contraindication during exercise. This study was approved by the Institutional Review Board of Zhejiang University and was carried out in compliance with the Declaration of Helsinki.Twenty-one healthy young adults from Zhejiang University were recruited in this study No-Break, Exercise-Break, and Rest-Break session, respectively), participants were required to perform a 30-min PVT with or without a break. The design of PVT was the same as in our previous studies  or cycling on a Monark 975 stationary exercise bicycle (namely mid-task exercise) in the middle of the tasks. Sessions were arranged at the same time of the day to eliminate the impact of circadian rhythm . In addition, subjective feelings of mental states were assessed via the Short Stress State Questionnaire (SSSQ)  according to the International 10–20 system during the 30-min PVT for three sessions. The sample rate was set at 2,000 Hz with reference to the FCz. The impedance was kept below 5 kΩ during the whole data collection. In the offline analysis, a previously validated standard EEG preprocessing pipeline was adopted. Briefly, all data were down-sampled to 256 Hz, bandpass filtered into 1–40 Hz, 50 Hz notch filtered, and average re-referenced. Independent component analysis (ICA)  was estimated as the ratio of the specific band power to the total power spectral that was estimated with a frequency range of 1–40 Hz. For each 3-min epoch, averaged relative power was obtained from all included trials.Each 6-s EEG data trial was divided into segments using a 100% Hamming window of 1 s, then the power spectral density (PSD) was estimated by Welch's method, and specially focused on two low-frequency bands: delta band (1–4 Hz) and theta band (4–7 Hz) for their potential sensitivity toward mental states  repeated measures two-way ANOVA on RT was adopted to explore the immediate influence of breaks, while another repeated measures two-way ANOVA with Time (T1 and T4) by Session  on RT was conducted to investigate the long-lasting general effect.Consistent with the previous study  repeated measures ANOVA was conducted on the SSSQ scores of each category.As mentioned previously, the subjective feelings of participants were also assessed No-Break, Exercise-Break, and Rest-Break). Specifically, the dependent variables were the multiband EEG power ratio in different brain clusters.To comprehensively depict the break-related spectral changes in the low-frequency range, averaged EEG power ratio of two frequency bands (delta and theta) in the three clusters  was obtaPδ, ΔPδ, Pθ, ΔPθ) to investigate the behavior–EEG relationship in the immediate effects and general effects in different sessions.For the frequency band displaying the significant spectral difference in the last step, the correlation analysis was conducted between the changes of RT  and relative EEG .Normality and sphericity of data were checked for statistical analysis. A value of No-Break session into 10 3-min bins to evaluate the trend of behavioral performance over the task  = 22.270, p < 0.001, η2 = 0.553] of reaction time was observed from T1 to T4. The individually fitted linear slope of the RT also showed a performance decline throughout the task (slope Mean ± SD = 0.022 ± 0.004), in which averaged RT was 11.78% longer during T4 (most fatigued) in comparison with T1 (most vigilant).To investigate the time-on-task (TOT) effect induced by PVT, we averaged the reaction time (RT) in the the task . SpecifiF = 4.942, p = 0.013, η2 = 0.215], time , and session . Following post-hoc analyses indicated that the significant interaction effect was attributed to the decrease of RT in both the Exercise-Break session  and the Rest-Break session . However, no significant difference was observed between the Exercise-Break session and Rest-Break session in their post-break RT, indicating a similar vigilance improvement immediately after different interventions.For the immediate effect (T2 vs. T3) of mid-task breaks , there wF = 65.451, p < 0.001, η2 = 0.784] and sessions , but no time-by-session interaction . Follow-up post-hoc comparisons for the main time effect suggested that RT in T4 was significantly longer than that in T1 . Specifically, compared with T1, RTs in Exercise-Break and Rest-Break sessions increased by 6.970 and 8.297%, respectively. In addition, post-hoc comparisons for the main session effect indicated that in the T4, the RT in the Exercise-Break session was significantly shorter than that in the No-Break session .The general effect (T1 vs. T4) of mid-task breaks was also tested . SignifiF = 21.535, p < 0.001, η2 = 0.545] and distress  scores, but no significant difference of session  and time-by-session  interaction. Further inspection of the main effect in time suggested that for No-Break and Exercise-Break session, participants showed significantly increased distress  as well as less engagement  in the post-session compared with pre-session, indicating a similar trend of subjective experience between successive PVT and exercise-inserted PVT.In terms of subjective states, significant main effect of time was found on both engagement  and PParietalθ_ . Following post-hoc analysis indicated that, in the Exercise-Break session, Pθ significantly decreased in T3 compared with T2, in PFrontalθ_ , PParietalθ_  in comparison with a maintained Pθ in Rest-Break and No-Break sessions (The immediate effect of mid-task breaks on the EEG power ratio was first assessed. We observed a significant difference in theta and delta activities in T3 (post-break bin) compared with T2 (pre-break bin). Specifically, in theta band, there were significant main effects for time-by-session interaction, in Psessions .PCentralδ_ . The post-hoc analysis indicated that, in the Exercise-Break session, PCentralδ_ significantly increased in T3 compared with T2   = 5.221, p = 0.010*, η2 = 0.225]. The post-hoc comparison showed a significant decrease in T4 compared to T1  in the Rest-Break session, as well as an increase trend  in the Exercise-Break session  with T1 (first 3-min bin). A significant main effect for time-by-session interaction was also observed in P session .δ and Pθ demonstrated significant differences immediately after the mid-task exercise, while Pδ also suggested a significant difference in the general effect of the Rest-Break session. Consistent with the interaction effect observed in EEG power, correlation analyses were mainly focused on the immediate effect of the Exercise-Break session and the general effect of the Rest-Break session. Correlation investigation between the EEG power and behavioral performance showed that Pθ in T2 was correlated with ΔRTT3−T2  in the exercise-inserted task, and Pδ in T3  was significantly related with ΔRTT4−T1 in the rest-intervention task. No significant association was observed between ΔPT3−T2θ and ΔRTT3−T2 , ΔPT3−T2δ and ΔRTT3−T2  in Exercise-Break session, as well as ΔPT4−T1δ and ΔRTT4−T1  in Rest-Break session. In particular, the EEG power in correlation analysis was acquired from global electrodes rather than specific clusters to preliminarily explore the biomarker of mental fatigue recovery  Correlation analysis suggested that the immediate improvement of behavioral performance  for exercise intervention was associated with theta power in T2. Interestingly, we found the general behavioral effect  in the Rest-Break session was correlated with delta power in T3. These findings are discussed in greater detail below.In this study, we revealed divergent post-break improvement neural patterns following mid-task exercise and rest break under similar beneficial effects on behavioral performance, and further investigated the neural basis underlying mental fatigue restoration. The significant findings are as follows: (1) behaviorally, we found a transiently restorative effect of both exercise and rest breaks on sustained attention, without any general beneficial effect on behavioral performance. (2) Exercise and rest demonstrated different patterns of EEG change in post-break improvement. In particular, theta decreased and delta increased immediately after the mid-task rest, this behavioral result was inconsistent with our early findings that no significant restorative effect was observed after rest breaks , the National Key Research and Development Program of China (Grant No. 2021ZD0200400), the Key Research and Development Program of Zhejiang Province (Grant No. 2022C03064), the Zhejiang University Global Partnership Fund (Grant No. 100000-11320), and the Hundred-Talent Program of Zhejiang University. This work was partially supported by the Shanghai Municipal Science and Technology Major Project (Grant No. 2021SHZDZX0100) and the Fundamental Research Funds for the Central Universities.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Bursaphelenchus xylophilus) to new areas has affected the international forestry industry because this pathogen causes pine wilt disease (PWD). Therefore, methods for the accurate and reliable detection of B. xylophilus are essential for controlling and managing this pest. The PCR and Loop-Mediated Isothermal Amplification (LAMP) techniques developed in this study involve species-specific primer sets targeting B. xylophilus genes encoding potential molecular mimicry proteins , which are associated with pathogenicity. The PCR and LAMP results revealed that the primers were specific for B. xylophilus Bx-tlp-1, Bx-tlp-2, and Bx-cpi. Moreover, our LAMP assay targeting Bx-tlp-1 conducted at 63°C detected B. xylophilus within 20 min and B. xylophilus from Monochamus alternatus or M. saltuarius within 30 min. The lower limits of detection for the LAMP and PCR assays were 10 pg and 10 ng genomic DNA, respectively, implying these assays may be useful for the rapid detection of B. xylophilus in pine forests. Designing primers specific for Bx-tlp-1, Bx-tlp-2, and Bx-cpi enabled the relatively rapid detection of B. xylophilus isolates as well as M. alternatus or M. saltuarius carrying B. xylophilus. These primers, which were designed following a thorough functional analysis of key B. xylophilus pathogenicity-related genes, may be useful for developing improved assays for the early diagnosis and prevention of PWD.The introduction of the pine wood nematode ( Bursaphelenchus xylophilus, in three steps . The amplified sequence serves as a template for the next cycle. Because each cycle takes 2–4 min to complete, the target sequence can be amplified several million times in 2–3 h. Because of it, the PCR technique is mature for clinical diagnoses and quarantine. The LAMP technique, which can be used to rapidly amplify DNA, requires four primers specific for six regions of the target DNA. The target sequence is then amplified by elopment .B. xylophilus. The proposed PCR and LAMP techniques use the species-specific primer sets targeting B. xylophilus genes encoding potential molecular mimicry proteins, including thaumatin-like protein-1 , thaumatin-like protein-2 , and a cysteine proteinase inhibitor . B. xylophilus thaumatin-like proteins (TLPs) and one cysteine proteinase inhibitor with sequences that were highly similar to those of plant proteins. These proteins were subsequently determined to induce cell death in Nicotiana benthamiana (B. xylophilus and Pinus massoniana are reportedly similar (P. massoniana TLP (Pm-tlp) and revealed that its expression is associated with the expression of the B. xylophilus Bx-tlp-1. The relatively high sequence similarity between potential molecular mimicry proteins and plant proteins suggests that they may have similar functions. An analysis of the expression of the potential molecular mimicry proteins in B. xylophilus infecting pine trees indicated that the temporal changes to the α-pinene content in the trees are consistent with the expression levels of the genes encoding a TLP (CPI) in B. xylophilus and P. massoniana. Thus, these genes are likely important for B. xylophilus infections of pine species  encoding previously identified potential molecular mimicry proteins  consisted of the following: PrimeSTAR HS DNA , 12.5 μl; forward and reverse primers, 1 μl each; DNA template, 1 μl; and ddHB. xylophilus DNA) and a negative control (distilled water) were included in each run. The LAMP amplicons were detected by fluorescence using LightCycler 480 II system  or by a color change in the reaction mixture under natural light.The LAMP mixture (25 μl) comprised the following: primers F3 and B3, 5 pmol each; primers FIP and BIP, 40 pmol each; primers Loop F and Loop B, 20 pmol each; 2 × Reaction Mix , 12.5 μl; and DNA template, 1 μl. Samples were incubated at 63°C for 60 min and then at 85°C for 3 min to terminate the reaction. A positive control  per reaction.The sensitivity for the PCR and LAMP primer sets  was assessed by performing the assays using a range of copy numbers  were amplified by PCR. The amplified Bx-tlp-1, Bx-tlp-2, and Bx-cpi fragments were 755, 241, and 202 bp, respectively . Accordingly, the PCR primers designed to target Bx-tlp-1, Bx-tlp-2, and Bx-cpi were specific for B. xylophilus. The LAMP primers targeting syg-2 designed by B. xylophilus isolates from Liaoning province , whereas the detection limit of the PCR assay for the same genes was 10 ng  may be more sensitive than the primers targeting syg-2.The results of the PCR amplification conducted to detect n 50 min . The prin . Therefore, the numbers of beetles  in a pine forest is a crucial indicator of the possibility of a PWD outbreak. Unless B. xylophilus from M. alternatus or M. saltuarius is specifically treated in the primary phase, the resulting PWD will often lead to tree death. The efficient and rapid detection of B. xylophilus from M. alternatus or M. saltuarius is necessary for preventing outbreaks via the implementation of specific treatments  is responsive to α-pinene, which may affect terpene metabolism in pine trees and influence the pathogenicity of B. xylophilus , and the lower limits of detection for the LAMP and PCR assays were 10 pg and 10 ng genomic DNA, respectively. Moreover, LAMP primers targeting Bx-tlp-1 or Bx-cpi were 5-fold more sensitive than the LAMP primers targeting syg-2  to minimize the possibility of contamination . The LAM for PCR .M. alternatus is the main insect vector for B. xylophilus. The number of nematodes in M. alternatus is an important factor related to the distribution of B. xylophilus. Therefore, the fast and efficient detection of M. alternatus carrying B. xylophilus is necessary for the monitoring and early detection of PWD . The specific amplified fragments for Bx-tlp-1, Bx-tlp-2, and Bx-cpi were 755, 241, and 202 bp, respectively. However, no specific amplification products were detected for the other analyzed nematodes . These findings indicate that Bx-tlp-1, Bx-tlp-2, and Bx-cpi can be used to specifically detect B. xylophilus as well as M. alternatus or M. saltuarius carrying B. xylophilus. Furthermore, designing primers specific for B. xylophilus following an in-depth analysis of the functions of key pathogenicity genes may have important implications for future attempts at developing reliable methods for the early diagnosis and prevention of PWD.In conclusion, n of PWD . In thisThe datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/FM, YL, and XZ: experimental design. FM and ZL: experimental implementation. ZL: material contribution. FM: data analysis and manuscript writing. All authors contributed to the article and approved the submitted version.The present research was funded by the Fundamental Research Funds for the Central Universities (2021ZY03) and China Postdoctoral Science Foundation (2020M680410).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


ORF1a and N) and an LoD for the ORF1a and N genes as 39 and 25 copies/reaction, respectively. It can be potentially used as a high-throughput national screening for different respiratory-based infections within the Middle East region, such as the MERS virus or major zoonotic pathogens such as Mycobacterium paratuberculosis and Brucella spp., particularly in remote and rural areas where lab equipment is limited.Since the COVID-19 pandemic outbreak in the world, many countries have searched for quick diagnostic tools to detect the virus. There are many ways to design diagnostic assays; however, each may have its limitations. A quick, sensitive, specific, and simple approach is essential for highly rapidly transmitted infections, such as SARS-CoV-2. This study aimed to develop a rapid and cost-effective diagnostic tool using a one-step Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP) approach. The results were observed using the naked eye within 30–60 min using turbidity or colorimetric analysis. The sensitivity, specificity, and lowest limit of detection (LoD) for SARS-CoV-2 RNA against the RT-LAMP assay were assessed. This assay was also verified and validated against commercial quantitative RT-PCR used by health authorities in Saudi Arabia. Furthermore, a quick and direct sampling from the saliva, or buccal cavity, was applied after simple modification, using proteinase K and heating at 98 °C for 5 min to avoid routine RNA extraction. This rapid single-tube diagnostic tool detected COVID-19 with an accuracy rate of 95% for both genes ( In late 2019, Wuhan in Hubei Province in China reported an unknown fast-spreading viral outbreak as the world’s fastest pandemic disease in the twenty-first century. The World Health Organization (WHO) declared it soon after as COVID-19 . In 2002Despite the efforts to develop antiviral drugs for COVID-19 and the global coverage of vaccination, the incidence rate is high in many countries, including the United States, where more than 85,007,630 infected cases have been detected, with 1,002,946 deaths. By 17 June 2022, SARS-CoV-2 infection had rapidly spread and had infected more than 535,863,950 people, with more than 6,314,972 deaths reported worldwide. In Saudi Arabia, there have been 781,168 infected cases and a total of 9179 deaths up to that date  .Considering the previously mentioned drawbacks, sample processing and management challenges for the frontline workers against COVID-19, the continuing fast spreading of infection, the time taken for each test, and the cost of testing, the development of a simple yet specific and rapid diagnostic tool is still desired. Thus, this study underpins Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP) for easy and fast diagnosis of COVID-19. LAMP is a quick, specific, sensitive, and cost-effective diagnostic technology that could replace current RT-PCR. This technology only needs a heat block or water bath to maintain a temperature of 65 °C to conduct a reaction. A DNA polymerase with chain displacement activity (Bst) is coupled with reverse transcription enzyme activity in one single tube that can be developed and optimized for rapid detection of COVID-19. The reaction results can be visualized after 30–60 min by observing either the turbidity or colorimetric analysis using the naked eyes or under the UV lamp ,12,13.N gene [Several studies have reported the successful use of LAMP for the rapid and specific detection of COVID-19. Lu et al. developed this simple and rapid assay for SARS-CoV-2 detection using the viral N gene . The resN gene . AnotherN gene . RoumaniN gene . AnastasN gene . These sORF1a/b and N genes. The findings indicated that this assay is affordable (≈1.7 €/sample), with a rapid detection of under 25 min [Furthermore, Diego and colleagues have successfully developed a rapid detection of SARS-CoV-2 targeting both r 25 min . A furthr 25 min . Other sr 25 min . Anotherr 25 min . Finallyr 25 min . The aboE, N, and RdRp genes was reported by Fellner et al. [E and N genes providing greater sensitivity, while all three genes can improve the specificity. The RT-LAMP assays showed specificity and sensitivity for the N + ORF1 genes as 100% and >75%, respectively, while the specificity and sensitivity were 83.9% and 100% for the E gene, respectively. This variability of the diagnostic assay performance suggested that selecting the correct gene can be a crucial step when developing the most appropriate diagnostic test [ORF1ab, N, and S). The results demonstrated that the RT-LAMP has a 100% concordance in both sensitivity and specificity with RT-PCR for detecting SARS-CoV-2 nucleic acid, with a reaction time of 30 min for the RT-LAMP assay [ORF1ab and N genes) in the Saudi population. In addition, a quick and direct sampling from the saliva, or buccal cavity, was applied after simple modification to avoid the routine and time-consuming RNA extraction method.Several studies have reported the successful use of LAMP for detecting SARS-CoV-2, yet it is still not widely used clinically in many countries. A study performed in Argentina evaluating several commercially available RT-qPCR and RT-LAMP assays against SARS-CoV-2 r et al. . The RT-tic test . In SaudMP assay . TherefoORF1a/b, which encodes for two big polyproteins with a size of 1888 bp. Another vital gene considered in this study and presented in the SARS-CoV-2 genome is the N gene, which encodes for structural protein “nucleocapsid phosphoprotein” with 1260 bp in size. These two targets have been recommended by the Chinese CDC and published lately [ORF1a and N gene) were synthesized in vitro by DNA Technologies, Inc. (IDT), Coralville, IA, USA. Positive controls were synthesized for the nucleocapsid gene  and partial ORF1 (510 bp). The LAMP technology uses six specific primers targeting eight different sequences on the gene of interest. Six specific LAMP primers were designed to target a small fragment of ORF1a, N genes and Ribonuclease P (RNAse P), as demonstrated in the https://primerexplorer.jp/e/ (accessed on 7 July 2020)).The total size of the SARS-CoV-2 genome is 30 kb in GenBank (Accession number: MN908947). The genome of the virus contains ten open reading frames (ORFs). The first and largest one is d lately . TherefoORF-1a and N), primers were designed to amplify 510 bp and 722 bp from ORF1a and N, respectively. T7 promoter sequence has been added to the forward primer (5′-TAATACGACTCACTATAGGGAGA-3′) to facilitate the RNA transcription. The PCR was performed in two-cycle conditions using SeqAmp DNA Polymerase . In the first 15-cycle, the target region is amplified, followed by 20-cycle to incorporate T7 in the amplicon. The forward primer for ORF-1a (5′-TAATACGA CTCACTATAGGGAGA-TTCAACCAAGGGTTGAAAAG-3′) and the reverse (5′-GTATTTCAAGAAGGTTGTCATT AAG-3′) while the forward primer for N (5′-TAATACGACTCACTATAGGGAGA-TGGACCCCAAAATCAGCG-3′) and the reverse (5′-TTTGGCCTTGTTGTTGTTG-3′) were used to amplify the target. The amplicons were purified using QIAquick PCR Purification Kit , followed by RNA transcription using a HiScribe™ T7 High Yield RNA Synthesis Kit , per the manufacturer protocol. The transcribed RNA was treated with 1 μL DNase I reagent (incubate at 37 °C for 10 min), followed by the purified step using Monarch RNA Cleanup Kit , and then quantified by the SpectraMax QuickDrop spectrophotometer .To synthesize RNA from the control DNA fragments (® colorimetric LAMP 2X Master Mix (DNA and RNA) . The reaction was designed to provide a one-step LAMP of RNA (RT-LAMP) targets. The kit contains a blend of Bst 2.0 WarmStart DNA Polymerase and WarmStart RTx Reverse Transcriptase in an optimized LAMP buffer solution. The reaction includes 2.5 μL of 10× primer mix , 12.5 μL of WarmStart LAMP 2X master mix, 1 μL of the sample and the reaction was brought up to 25 μL by doubled distilled water . The reaction was then vortexed and collected again by quick centrifugation before it was sealed and placed in a heat block at 65 °C for 30–60 min. The reaction moved to PCR Cooler Rack (or ice) for 5–10 s and then observed visually against the contained pH indicator. The pink color signifies a negative reaction, while the yellow color confirms a positive reaction.The colorimetric RT-LAMP assay was performed in 25 μL reaction volume using WarmStartDifferent RNA samples were used to validate and test the developed RT-LAMP assay, including clinically isolated RNA samples. The validation was carried out using blind clinical samples, i.e., nasopharyngeal swabs, to test if the new assay concordant with the RT-PCR commercial kit results. This validation study was performed by the Saudi CDC , Riyadh, Saudi Arabia, after obtaining an ethical approval number of H1RI-15-Nov20-01 from the King Saud Medical City (KSMC) institutional review board (IRB), Riyadh, Saudi Arabia, for using patients’ biological samples. Different clinical samples (3 μL each) were run using RT-PCR and RT-LAMP side by side to confirm the validation of the latter tool. The level of sensitivity, specificity, and reproducibility was estimated accordingly. The LoD in RNA copy number, positive predictive value (PPV), and negative predictive value (NPV) were computed to validate the developed RT-LAMP assay.The RNA extraction from saliva and the buccal swab was completed by transferring 500 μL of phosphate normal saline (PBS) pH 7.4  to the samples before RNA extraction. A 50 μL sample was added to an equivalent volume of PBS (50 μL) alone or with 5 μL proteinase K . In addition, 1M Tris-HCl buffer with a pH 8.0  was used as an alternative buffer to PBS. Furthermore, RNA from the samples extracted by Monarch Total RNA. Miniprep Kit  according to the manufacturer’s protocol.ORF1a and N, in the SARS-CoV-2 virus (ORF1a and N genes gave positive results (yellow color) with 9 × 109 and 2 × 109 DNA copies, respectively. The color for the control sample, i.e., with no DNA template, remained pink . The five targets related to the N gene were evaluated with similar primers to the previous studies [ORF1a was explored as a potential target with RT-LAMP assay [ORF1a gene were assessed in this study, which was not reported previously. This assay evaluated the primer’s specificity in the B region in silico against SARS-CoV-2 or SARS-CoV-1 on the same targeted genes. The B target on the ORF1a showed more specificity for SARS-CoV-2 in terms of mismatching with a similar coronavirus (SARS-CoV-1) with 54 mismatches from 204 bases. On the other hand, the B target on the N gene exhibited a more similarity to SARS-CoV-1, with 20 mismatches from 213 bases showing less specific primer binding . The primer targeting the B region in both genes demonstrated the highest sensitivity for the DNA and RNA. Ninety-six clinical samples  were tested to validate this RT-LAMP assay on actual clinical samples. All samples were positive for SARS-CoV-2 using RT-PCR assay with quantification cycle (Cq) values ranging from 14 to 34, as presented in The previous test aimed to determine the most sensitive target primer candidate in both genes , in which the results did not pick up by the RT-LAMP assay or took a longer time > 35 min to be detected, which could affect some negative samples and might cause false-positive results ,33,34. OBst DNA polymerase activity [The RNA extraction in the clinic setting required kits and particular types of machinery, which were not compatible with the simplicity of the RT-LAMP assay. To optimize the RNA extraction, the RNAse P is used regularly in SARS-CoV-2 diagnosis as an internal control. To address that, different methods for RNA extraction using two variants, i.e., temperature and proteinase K, on two types of samples: buccal swab and saliva. First, the sample was added directly to the RT-LAMP reaction to confirm whether the temperature (65 °C in 30 min) could trigger the RNA’s release and amplify the target (RNAse P). However, this outcome was not achieved from saliva or buccal swab A3, whichactivity ,37. InteThe minimum time required for the RNA release with proteinase K and heat was furtherly assessed. A low temperature of 55 °C for 5 min with proteinase K yielded a negative reaction  for the buccal swab and a positive reaction  for the saliva sample A1,B1. By2 viral copies by LAMP assay [RNA extraction is vital for assays based on detecting nucleic acids . Hence, MP assay . AnotherMP assay . The comMP assay ,41,42.ORF1a and N genes . Unexplored regions in the ORF1a and N genes with novel primer sites have been identified. The primer design is a crucial step for nucleic acid amplification. Employing loop primers increased the assay specificity and reduced the reaction time. Furthermore, not all targets in ORF1a and N genes gave the same sensitivity, which may be due to the nature of the target gene or primer design factors. This assay detected a two-digit copy number of the viral RNA expressing its high sensitivity level. The developed RT-LAMP assay was validated against 96 positive RNA clinical samples, yet various clinical validations to ensure that this assay operates under different testing conditions are needed. Clinical validation with RT-LAMP demonstrated approximately 95% for both genes (ORF1a and N), and the sensitivity dropped when tested against samples with Cq > 30 values, which would require the assay for further optimization. However, this drop in sensitivity was also reported in previous studies. Currently, the sample to RT-LAMP assay was experimented with and validated. There is still an attempt to use a more straightforward RNA extraction method without additional techniques or equipment to ease the process of COVID-19 and other respiratory-based viral infection detection. Therefore, two RNA extraction methods for two types of samples  were investigated. It showed that using a heat treatment at 98 °C with proteinase K can result in a positive reaction (yellow color) of RNA release in 5 min only for both sample types. This rapid single-tube diagnostic tool was able to detect SARS-CoV-2. It can potentially be used as a high-throughput national screening for different respiratory-based infections within the Middle East region, such as the MERS virus or Brucella bacterium, particularly in remote and rural areas where lab equipment is limited. However, extensive clinical validation for the developed RT-LAMP is required as an essential step in the potential commercialization of this assay.With the continuation of high COVID-19 transmission worldwide, there is still a need to constantly develop preventive or therapeutic tools and approaches to eradicate this pandemic. With the difficulty of delivering vaccines to countries with low incomes and poor healthcare services, there is still a gap in overcoming such diseases. Therefore, developing a diagnostic tool for SARS-CoV-2 with numerous advantages compared to other available virus detection assays, including its simplicity, rapidity, cost-effectiveness, sensitivity, and specificity, is needed. The developed RT-LAMP assay in this study has great potential to fulfill those criteria. The developed RL-LAMP assay showed high sensitivity in a short reaction time. The initial evaluation of five targets in


P < 0.001). The absolute value of the measured difference in mean vertebral height between the VTS and 1-mm-thick images was smaller than that between the 4-mm-thick and 1-mm-thick images (P < 0.01–0.54). The diagnostic performance of the VTS images for detecting compression fracture was significantly lower than that of the 4-mm-thick images for one reader (P = 0.02). VTS technique enabled the identification of each vertebral body, and enabled accurate measurement of vertebral height. However, this technique is not suitable for diagnosing compression fractures.Virtual thin-slice (VTS) technique is a generative adversarial network-based algorithm that can generate virtual 1-mm-thick CT images from images of 3–10-mm thickness. We evaluated the performance of VTS technique for assessment of the spine. VTS was applied to 4-mm-thick CT images of 73 patients, and the visibility of intervertebral spaces was evaluated on the 4-mm-thick and VTS images. The heights of vertebrae measured on sagittal images reconstructed from the 4-mm-thick images and VTS images were compared with those measured on images reconstructed from 1-mm-thick images. Diagnostic performance for the detection of compression fractures was also compared. The intervertebral spaces were significantly more visible on the VTS images than on the 4-mm-thick images ( Sagittal reformatted images are useful for evaluating spinal lesions and morphology3, for numbering the vertebrae, and identifying the vertebral level of lesions. However, it is not possible to reconstruct thin-slice images after the raw data have been deleted from storage in the scanner. Although saving thin-slice images for all patients before deleting the raw data would be one way of solving this problem, it would require more storage in picture archiving and communication system (PACS), which is costly.Multidetector-row CT (MDCT) is currently in widespread use worldwide, and can reconstruct thin-slice images with a slice thickness of 1 mm or less. Thin-slice images are useful for evaluating small lesions and depicting detailed structures, and are also used for reconstructing multiplanar reformatted images and 3-dimensional (3D) images using processes such as volume rendering and maximum intensity projection7. Generative adversarial networks (GANs) are deep learning models that comprise two networks trained simultaneously: a generator and a discriminator, and are aimed at generating new images8. GANs are being increasingly employed for various radiology applications, such as in noise reduction of CT images and for generating images of different modalities13. Virtual thin-slice (VTS) technique is a newly developed GAN-based algorithm that can generate 1-mm-thick virtual images from CT images obtained with a slice thickness of 3–10 mm14. This software is now commercially available and can be applied to CT images obtained with any CT scanner. If VTS images are as useful as true thin-slice images, then thin-slice images would not need to be prepared in advance and could be created when needed. It would also make it possible to create thin-slice images for comparison, even when thin-slice images have not been saved in previous examinations. However, it is necessary to verify the diagnostic ability of VTS images before this technique can be applied to clinical practice. Although many studies have reported use of the GAN technique for creating medical images13, few have examined whether the created images have the same diagnostic ability as the true images. The purpose of this study was to investigate whether VTS images can be used as a substitute for true thin-slice images in evaluation of the spine by analyzing the visibility of the vertebral bodies, accuracy of vertebral body height measurement, and diagnostic performance in detecting compression fractures.Artificial intelligence (AI) has been attracting greater attention in recent years and many reports regarding the use of AI in radiological image analysis have been publishedThis retrospective study was approved by the Osaka University Clinical Research Review Committee, and the requirement for informed consent was waived by the Osaka University Clinical Research Review Committee. All methods were carried out in accordance with relevant guidelines and regulations. Patients who underwent CT for evaluation of aortic or cardiac disease were eligible for inclusion in this study because we obtained a single scan in one breath-hold from the supraclavicular area to the symphysis pubis in these patients, whereas separate scans were obtained for the chest and abdominopelvic regions in other patients. Enrolled were 73 consecutive patients who underwent CT between January and February 2019 or between December 2020 and January 2021 . The clinical indications for CT in these patients are listed in Table CT was performed using a 160- or 320-slice CT scanner . A pre-contrast scan was performed in all patients from the supraclavicular area to the symphysis pubis during a single breath hold. Tube current was adjusted individually using an auto-exposure control technique with a standard deviation setting of 15. The remaining scan parameters were as follows: tube voltage, 120 kVp; rotation time, 0.5 s; helical pitch, 0.83. Although post-contrast scans were also acquired in 31 patients, only the pre-contrast images were used in this study.From the raw data of each patient, two sets of axial images were reconstructed, with a slice thickness/interval of 4/4 and 1/1 mm. A hybrid iterative reconstruction algorithm  with a weak strength setting was applied. The remaining reconstruction parameters were as follows: kernel, FC03; reconstruction field of view, 350 mm .14. VTS software was applied to the 4-mm-thick data set of each patient to generate 1-mm-thick VTS images.VTS is a conditional-GAN based algorithm. Thick-slice images with slice thickness/intervals of 3–10 mm were randomly simulated from real thin-slice images by down-sampling with Gaussian smoothing. A pair of original thin-slice images and simulated thick-slice images were used to train the VTS generator in the GAN framework  independently reviewed the sagittal images reformatted from 4-mm-thick images and the VTS images and evaluated the visibility of the intervertebral spaces in each of four regions: cervical, upper thoracic, lower thoracic, and lumbar spine. They reviewed these images on a commercially available workstation , and assigned a score using the following 4-point scale: 4, all intervertebral spaces are visible; 3, most intervertebral spaces are visible but some are unclear; 2, most intervertebral spaces are unclear; 1, no intervertebral spaces are visible. The radiologists were informed that the images for evaluation were either 4-mm-thick or VTS images, but were blinded to the patients’ identity, medical background, and the reconstruction protocol used.1) were calculated, as well as the absolute values of the difference between the measured heights on VTS and true 1-mm-thick images (D2). The absolute percentage errors between the measured heights on the 4-mm-thick and true 1-mm-thick images (%Error1) was also calculated by dividing D1 by the measured height on true 1-mm-thick images, as well as the absolute percentage errors between the measured heights on VTS and true 1-mm-thick images (%Error2). Measurements were performed using a workstation (SYNAPSE VINCENT version 5.3.001).Two radiologists familiar with abdominal radiology (16 and 9 years’ experience), different to the radiologists who performed the qualitative assessment, independently measured the height of the first thoracic (Th1) and first lumbar (L1) vertebrae on sagittal reformatted images made from each of the 4-mm-thick, true 1-mm-thick, and VTS data sets. Height was measured at the anterior border of each of these vertebrae. The absolute values of the difference between the measured heights on the 4-mm-thick and true 1-mm-thick images (D15. The reference standard was determined by two other radiologists (16 and 9 years’ experience) who evaluated the presence or absence of compression fracture on sagittal images reformatted from the true 1-mm-thick images, in consensus.The same two radiologists who performed the qualitative assessment also independently evaluated the possible presence of compression fracture using the sagittal reformatted images constructed from each of the 4-mm-thick images and the VTS images. They classified the likelihood of compression fracture in all vertebrae using the following 4-point confidence score scale: 1, probably no fracture present; 2, indefinite presence of fracture; 3, fracture probably present; and 4, fracture definitely present. Before the assessment, they were informed that a confidence level of 3 or 4 would be considered a positive finding for the calculation of sensitivity and positive predictive value (PPV). The criteria for compression fracture used in this study were: 1, ratio of the anterior height of the vertebra (AH) to the posterior height (PH) < 0.75; 2, ratio of the central height of the vertebrae (CH) to AH or PH < 0.8; 3, height of a vertebra reduced by > 20% compared with those above and below1 and D2) were compared using paired t-test. The absolute percentage errors of the measured vertebral heights (%Error1 and %Error2) were also compared using paired t-test. Interobserver agreement for each of D1 and D2 was evaluated by intraclass correlation coefficient (ICC). To analyze diagnostic performance for detecting compression fracture, jackknife free-response receiver-operating characteristic (JAFROC) analysis was performed using JAFROC software . This software computes the figure of merit (FOM), which is defined as the probability that a lesion is rated higher than the highest rated non-lesion on a normal image16. In the present study, JAFROC1 was used rather than JAFROC or JAFROC2 because of its high statistical power for human observers17. For all tests, a P value less than 0.05 was considered significant.Visual scores regarding the visibility of intervertebral spaces were compared using Wilcoxon signed rank test. The absolute values of the difference in measured vertebral heights (DP < 0.001)  Fig. .Table 2M2) was less than that between 4-mm-thick and true 1-mm-thick images (D1) for both readers, and the difference was significant for L1 measured by Reader 1 (P < 0.01). The mean absolute percentage error of the measured height between VTS and true 1-mm-thick images (%Error2) was smaller than that between 4-mm-thick and true 1-mm-thick images (%Error1) for both readers, and the difference was significant for L1 measured by Reader 1 (P < 0.01). The ICCs of the two readers for height measured on the 4-mm-thick images were 0.461 and 0.795 for Th1 and L1, respectively, whereas those measured on VTS were 0.524 and 0.813 for Th1 and L1, respectively.The mean measured heights of Th1 and L1 are summarized in Table P = 0.02) , but visibility was improved on reformatted images made from VTS images . Thus, reformatted images made from VTS images would make it easier to recognize and number individual vertebral bodies, and thus identify the vertebral level of a lesion. Moreover, this technique would improve the quality of reformatted or 3D images, and it would make it easier to obtain an overview of the whole spine.GAN is a type of deep learning model capable of generating realistic-looking fake imagesThe absolute values of the differences in measured heights of thoracic and lumbar vertebrae between VTS and true 1-mm-thick images were smaller than those between 4-mm-thick and true 1-mm-thick images, and interobserver agreement was slightly improved using VTS compared with 4-mm-thick images. Therefore, reformatted VTS images might have the potential to achieve more accurate measurement of bone structures compared with reformatted images using 4-mm-thick images. However, the height tends to be underestimated when using VTS, and it remains unclear whether VTS can be used for quantitative evaluation instead of thin-slice images. Further studies will be necessary to confirm the usefulness of VTS images in quantitative evaluation.Regarding diagnostic ability for compression fracture, VTS had impaired performance compared with 4-mm-thick images, for the reason that slight compression fractures were sometimes not depicted correctly by VTS images Fig. . If loca14, it was considered that images with a thickness of 4 mm were the most suitable for this software. Third, the reference standard for compression fractures was determined by consensus reading of the true 1-mm-thick CT images, and other diagnostic modalities such as MR imaging were not performed. As the purpose of this study was to investigate whether VTS images could be used as a substitute for true thin-slice images, it was appropriate to use the thin-slice images as the gold standard.Our study had several limitations. First, this was a retrospective study, and the number of patients was relatively small. Second, although thick-slice images of 3–10-mm thickness can be applied to VTS software, we evaluated only 4-mm-thick images. Because thick-slice images with thickness 4 or 8 times that of the thin-slice images were used when training the VTSVirtual thin-slice technique enabled the identification of all vertebral bodies and more accurate measurement of vertebral height compared with thick-slice images, but is not suitable for the detection of compression fractures. Further improvements are needed before virtual thin-slice images can achieve the same diagnostic performance as true thin-slice images for detecting lesions.


Osteosarcoma (OS) is the most common primary bone malignancy in children and adolescents. microRNAs have been found to play a vital role in tumor angiogenesis. Here, we investigated the effects of miR-199a-5p on tumor growth and angiogenesis in osteosarcoma. Furthermore, the underlying molecular mechanisms and signaling pathways were explored.in vivo xenograft models were established to further investigate the intrinsic role of miR-199a-5p in osteosarcoma tumorigenesis and angiogenesis.The datasets were extracted from the Gene Expression Omnibus and the differentially expressed miRNAs (DEmiRNAs) were screened out by the GEO2R online platform. The potential target genes were predicted using the miRTarBase database. The predicted target genes were further analyzed by Gene Ontology and pathway enrichment analysis and a regulatory network of DEmiRNAs and their target genes was constructed. In addition, the effects of osteosarcoma cell derived exosomal miR-199a-5p on the proliferation, migration and neovascularization of HUVECs were evaluated by conducting EdU assays, Transwell experiments and tube formation assays. A dual-luciferase reporter assay was performed to detect whether VEGFA was the direct target of miR-199a-5p. Furthermore, in vivo results verified that overexpression of miR-199a-5p in osteosarcoma cells could suppress the growth and angiogenesis of tumors.A total of 149 DE-miRNAs were screened out, including 136 upregulated miRNAs and 13 downregulated miRNAs in human osteosarcoma plasma samples compared with normal plasma samples. A total of 1313 target genes of the top three upregulated and downregulated miRNAs were predicted. In the PPI network, the top 10 hub nodes with higher degrees were identified as hub genes, such as TP53 and VEGFA. By constructing the miRNA-hub gene network, we found that most of hub genes could be potentially modulated by miR-663a, miR-199a-5p and miR-223-3p. In addition, we found that the expression level of miR-199a-5p in exosomes derived from osteosarcoma cells was remarkably higher than the osteosarcoma cells, and the exosomes derived from osteosarcoma cells were transported to HUVECs. Overexpression of miR-199a-5p could significantly inhibited HUVEC proliferation, migration and neovascularization, whereas downregulation of miR-199a-5p expression exerted the opposite effect. Moreover, the Our results demonstrated that miR-199a-5p could be transported from osteosarcoma cells to HUVECs through exosomes, subsequently targeting VEGFA and inhibiting the growth and angiogenesis of osteosarcoma. Therefore, miR-199a-5p may act as a biomarker in the diagnosis and treatment of osteosarcoma. MiRNAs n vitro . It is wn vitro . Emerginn vitro , 11.Studies have shown that inhibiting the expression of miR-199a-5p can promote the invasion of human hepatocellular carcinoma . MiR-199in vitro and in vivo. In addition, miR-199a-5p inhibits transduction of the HIF-1α/VEGF signaling pathway. Taken together, our findings provide valuable clues to the pathogenesis of osteosarcoma and provide a promising target for the development of effective treatment in the future.In this study, we screened the differentially expressed miRNAs in osteosarcoma using the miRNA expression profile of GSE65071. Differential expression of miRNA target genes and their potential functions were predicted by functional enrichment and pathway enrichment analysis. Then, we constructed regulatory networks of osteosarcoma-related miRNAs and their target genes. We further verified the mechanism of miR-199a-5p and its target genes in the angiogenesis of osteosarcoma. HUVEC proliferation, migration, invasion and new vessel formation are significantly inhibited by miR-199a-5p both https://www.ncbi.nlm. nih.gov/geo/). GEO2R (http://www.ncbi.nlm.nih.gov/geo/geo2r), a web-based application, contributes to filtrating differentially expressed miRNAs (DEmiRs) through Bioconductor’s GEO Query and limma R software (http://mirtarbase.cuhk.edu.cn/). Enrichment analysis of biological processes, molecular functions and cellular components of target genes was carried out online through the Enrichr website (http://amp.pharm.mssm.edu/Enrichr/).The miRNA dataset GSE65071  was downsoftware . Therefosoftware  (http://https://string-db.org/cgi/input.pl?sessionId=mQ0AB12ssIaK&input_page_show_search=on). The interaction network diagram of “miRNA-mRNA” was constructed by Cytoscape software (Version 3.7.1). The differences in the expression of target genes in osteosarcoma and normal tissue were calculated by using the R2 database (https://hgserver1.amc.nl/cgi-bin/r2/main.cgi) (Mixed Osteosarcoma - Aqeilan - 18 - MAS5.0 - u133p2). The prognostic analysis of target genes in osteosarcoma were calculated via R2 database (https://hgserver1.amc.nl/cgi-bin/r2/main.cgi)  - Kuijjer - 127 - vst - ilmnhwg6v2). Set the threshold to Cox P value <0.05. The correlation coefficient was calculated using Spearman’s method.Online analysis was performed for the interaction of downstream target proteins through the STRING website  were purchased from Zhong Qiao Xin Zhou Biotechnology Co., Ltd. . HUVECs were cultured in endothelial cell medium containing 10% FBS, endothelial cell growth supplement and 1% double antibiotics . Both ceExosomes were isolated from the supernatant of osteosarcoma cells according to the previously described protocol . HOS celAccording to the manufacturer’s instructions, the osteosarcoma cell-derived exosomes were labeled with PKH26 . In short, the exosomes were incubated with PKH26 for 10 minutes at room temperature. The labeled exosomes were washed twice in PBS, centrifuged, resuspended in low serum medium, and incubated with HUVECs at 4°C for 4 hours. HUVECs were stained with phalloidin , which binds F-actin with high affinity. Cell nuclei were stained with DAPI  and analyzed by confocal microscopy.In Vitro Kit  was used to detect cells. The image was analyzed by ImageJ software.To examine the proliferation ability of HUVECs, the cells were seeded in 24 well medium containing 10% FBS for 24 hours. Then, the cells were cultured in serum-free medium, and mimics or inhibitors were added to the experimental group. After that, EdU labeling reagent  was added at a dilution of 1:1000. After 6 hours, the cells were fixed with paraformaldehyde for 30 minutes, and subsequently immersed in 2 mg/ml glycine solution for 5 minutes, and then incubated with 0.5% TritonX-100 in PBS at room temperature for 20 minutes. According to the instructions, a Cell-Light EdU Apollo567 4 HUVECs were added to the plate. The control group was treated with medium only, and the test group was mixed with 100 μL of exosomes. The total length of tube branches and tubes was calculated using ImageJ software.After dissolving the basement membrane matrix  at 4°C, it was infused into a 48-well plate at 100 μL per well and incubated at 37°C for 1 hour. Then, 200 μL of endothelial cell culture medium without FBS containing 2×10Western blotting was performed as previously described . Briefly4 cells) treated with miR-199a-5p mimics or inhibitor for 24 hours were washed and resuspended in RPMI 1640 medium supplemented with 0.1% BSA. The cells were then placed on the upper part of a 24-well Transwell insert coated with Matrigel® , while the lower chamber contained endothelial cell medium . After 6 hours, the filter was removed, fixed and stained with crystal violet staining solution .According to the manufacturer’s instructions, 8-μm pore filters were used for Transwell invasion assays  to determine the invasion. In brief, HUVECs  were subcutaneously injected in the right flank using HOS cells with stable expression of lentivirus miR-199a-5p with stable expression of miR-199a-5p or HOS cells with lenti-miR-199a-5p-NC or HOS cells with PBS. Mice were humanely sacrificed on Day 28. Tumor samples were weighed and then processed for routine immunohistochemistry and western blot  was applied for statistical analysis in this study. All experiments were repeated at least three times. All values are expressed as the mean ± standard deviation. Analysis of variance was used for comparisons between multiple groups. Student’s t-test was used to compare two independent groups. For the data conforming to the normal distribution, we use the two way t-test for statistical analysis. A P value of <0.05 was considered statistically significant.The GSE65071 dataset  was first downloaded and preprocessed  , molecular function (MF) and cellular component (CC). As shown in To explore the interactions of the target genes, a PPI network based on the STRING database was constructed. The results showed that many of these target genes could interact with each other. The top ten hub genes listed in Subsequently, Cytoscape software was used to construct the miRNA-hub gene network, as shown in https://hgserver1.amc.nl/cgi-bin/r2/main.cgi)  . As showhttps://hgserver1.amc.nl/cgi-bin/r2/main.cgi)   as shownTo evaluate the effect of osteosarcoma exosomes on tumor angiogenesis, we first needed to determine whether the tumor exosomes were taken up by HUVECs. The extracted exosomes of HOS cells were characterized using TEM, NanoSight and western blotting. TEM images showed that the majority of the particles exhibited a cup- or round-shaped morphology  also participates in the process of angiogenesis. After HUVECs were transfected with mimics or inhibitors, CD31 immunohistochemistry was performed. The results suggested that miR-199a-5p mimics obviously reduced the expression of CD31 in HUVECs, while the inhibitor distinctly enhanced the expression of CD31  group, as shown in in vivo.To further investigate the intrinsic role of miR-199a-5p in osteosarcoma tumorigenesis and angiogenesis, in vivo, H&E staining was used to detect the number of vascular tissues in tumor sections. The results indicated that the number of tumor vascular tissues in the Lenti-miR-199a-5p group decreased  , China postdoctoral science foundation (Grant number 2018M642668), and Natural Science Foundation of Shandong Province (ZR2021MH293).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


COVID-19 is associated with an increased mortality in hemodialysis patients. Therefore, achieving a long-lasting effective immune response to SARS-CoV-2 vaccines is essential. This study describes the humoral immune response in hemodialysis patients following three doses of mRNA vaccines against SARS-CoV-2, and explores the factors associated with a sustained immune response.We analyzed the monthly serological evolution of SARS-CoV-2 anti-S(RBD) antibodies for 1 year in 178 chronic hemodialysis patients who received three doses of SARS-CoV-2 mRNA vaccines. The primary outcome was sustained effective humoral response defined as anti-S(RBD) levels > 1,000 AU/ml after 4 months from the third dose. Multivariate logistic regression analyses were used to identify features associated with a sustained humoral immune response.After the initial two SARS-CoV-2 mRNA vaccine doses, 77.8% of patients showed an immediate effective humoral response, decreasing to 52.5% after 4 months. Antibody levels were significantly higher in COVID-exposed patients and HBV vaccine responders. After the third dose, 97% of patients showed an effective humoral response, and remained in 91.7% after 4 months. The mean monthly rate of antibody titer decline decreased from 33 ± 14.5 to 25 ± 16.7%. Multivariate regression analysis showed that previous exposure to COVID-19 and response to HBV vaccines were associated with an effective sustained humoral immune response.Immunization with SARS-CoV-2 mRNA vaccines elicits an effective immediate humoral immune response in hemodialysis patients, with a progressive waning in antibody levels. A third booster dose enhances the immune response with significantly higher antibody levels and more sustained humoral immune response. COVID-naïve patients and patients without previous response to HBV vaccines are likely to benefit from receiving more booster doses to maintain an effective immune response. Patients on hemodialysis (HD) have an increased incidence of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) compared to the general population, and when infected their mortality risk is higher –3. For tThere are no specific recommendations concerning the most adequate vaccine regimen for HD patients. Around 90% of HD patients without previous infection with SARS-CoV-2 develop an immediate humoral and cellular immune response after the administration of two doses of mRNA vaccines , 11. DesThere are no longitudinal studies concerning SARS-CoV-2 vaccine-induced immune responses for more than 4 weeks after receiving the third mRNA vaccine dose in patients on HD, which are needed to optimize clinical care and plan preventive strategies in this population.In this study, we describe the serological monthly changes against SARS-CoV-2 in HD patients after 1 year of follow-up from an initial immunization with two doses of mRNA vaccines. A third booster dose was administered after 5 months from the second dose, with continuous monthly serological assessment for 4 months following the third vaccine dose. We aimed to describe the factors associated with a sustained humoral response and identify the group of patients who are more likely to benefit from receiving a third booster dose.We designed an observational, longitudinal study to evaluate the immune response induced by the different mRNA vaccines against SARS-CoV-2 in patients attending two HD units: an in-center hospital dialysis unit and its affiliated satellite dialysis unit. The study comprised all HD patients who received vaccination against SARS-CoV-2 with a two-dose mRNA vaccine between December 28th 2020 and June 30th 2021, either BNT162b2 (BionTech/Pfizer) or mRNA-1273 (Moderna), given according to the manufacturer’s recommendations. The type of vaccine was assigned according to the available vaccines at the local vaccination point or at our center.Patients were classified as having had a prior COVID-19 infection if there was clear medical documentation with a positive SARS-CoV-2 PCR swab or in presence of nucleocapsid-IgG specific antibodies in serological analyses before the administration of the first dose of the vaccine. Patients who had received the vaccination schedule before starting HD, patients with an unknown serological status for SARS-CoV-2 before vaccination and patients who either received an adenoviral vector-based vaccine or did not complete the full SARS-CoV-2 mRNA vaccine schedule were excluded from the study.The study was conducted according to the guidelines of the Declaration of Helsinki and received the approval of the Ethical Committee for Clinical Investigation of the Hospital Universitario Fundación Alcorcón. All patients signed an informed consent form and approved the use of their anonymized clinical information for medical research purposes.Patients received two initial doses of either mRNA vaccine (Pfizer or Moderna) separated by 4 weeks, followed by a third booster dose after 5 months. Serum samples for SARS-CoV-2 anti-spike 1 receptor binding domain [anti-S(RBD)] IgG antibody titers were obtained 2 months prior to the administration of the first dose of the vaccine as well as immediately before vaccination, immediately after the administration of the first and the second dose of vaccine, and thence in a monthly basis up to 4 months after the third booster dose, which would correspond to 10 months after the administration of the first dose . This seWe examined associations between anti-S(RBD) IgG antibodies and potential predictors such as demographic and clinical data, type of dialysis and type of the vaccine, previous exposure to COVID-19, history of effective humoral response to hepatitis B virus (HBV) vaccines, and treatment with immunosuppressants. Medical histories of study participants were extracted from their medical records.Antibody response was determined using the chemiluminescent microparticle immunoassay of IgG antibodies to SARS-CoV-2 in human serum  which provides quantitative measures of IgG antibodies specific for SARS-CoV-2 S1-RBD. Results are given as arbitrary units per milliliter (AU/ml), and were interpreted as positive according to the manufacturer’s instructions, with a cutoff index value of > 50 AU/ml. The upper limit of quantification was 40,000 AU/ml. The humoral response was considered as effective when the value of SARS-CoV-2 anti-S(RBD) antibodies was > 1,000 AU/ml, which strongly correlates with a sustained neutralizing antibody response of 1/80 measured by plaque reduction neutralization titer , 19. TheDialysis vintage was defined as the time between the first HD session and the administration of the first dose of mRNA vaccine. Response to HBV vaccine was defined as an anti-HBs antibody titer > 10 IU/ml after four doses of Engerix-B or Fendrix. Immediate humoral response to SARS-CoV-2 vaccines was defined as an anti-S(RBD) antibody titer > 50 AU/ml 4 weeks after the second dose of the vaccine, and the same cutoff was used in every monthly serological assay. Effective humoral response was defined as an anti-S(RBD) antibody titer > 1,000 AU/ml, and sustained humoral response was defined as a persistent antibody titer > 1,000 AU/ml for more than 4 months.t-test, if normally distributed, or by the Mann-Whitney test if abnormally distributed. A Chi-square test was used for categorical variables. Multivariable logistic stepwise regression was used to determine the independent factors associated with an effective humoral response  4 months after the first two mRNA doses and 4 months after the third dose. The covariates of interest were selected based on the associations with serological response in univariate analyses, and any variables that were at the significance level p less than 0.10 in univariate analyses were included in these models. P-value of < 0.05 was considered statistically significant. Statistical analysis was performed with IBM SPSS Statistics for Windows, Version 22.0 .Demographic and clinical information are described for all patients included in the study. Data were presented as a number (percentage) for categorical variables, as mean ± standard deviation for continuous variables that were normally distributed, and as median  for continuous variables that were non-parametric. Continuous variables were first tested for normal distribution using Shapiro-Wilk, and then compared by One hundred and ninety-six patients on maintenance HD were screened for this study, 18 patients did not meet the inclusion criteria and were therefore excluded. Ultimately, 178 patients were eligible and were included in the study. p < 0.001). After receiving the third mRNA vaccine dose, the proportion of patients who seroconverted increased to 98.3%, and this rate remained stable for the following 4 months. Median antibody levels rose up to 26,037 AU/ml  and decreased to 9,180 AU/ml  after 4 months from the booster dose (p < 0.001).The evolution of IgG anti-S(RBD) antibody levels in the whole cohort throughout the study period is represented in p < 0.001].The proportion of patients with IgG anti-S(RBD) antibody levels over 1,000 AU/ml changed in a similar manner, from 77.8% of patients after receiving the first two doses, to 52.5% of patients 4 months after the second dose, rising up to 97% after receiving the third booster dose, and remaining in 91.7% of patients after 4 months from the third dose . Overalln = 33) and those who were COVID-19 naïve at the time of vaccination (n = 145) (p = 0.04), and lower proportion of patients with online haemodiafiltration  in the group of patients with prior COVID-19 infection. Pre-vaccination levels of anti-S(RBD) IgG antibodies were elevated in 78.8% of COVID-exposed patients, with a median antibody titer of 1,123 AU/ml, of which 51.5% had antibody levels higher than 1,000 AU/ml before vaccination. On the other hand, in the COVID-naïve group only two patients (1.4%) had positive anti-S(RBD) antibody levels that were slightly elevated (56 and 63 AU/ml) with negative IgG nucleocapsid antibodies and no history of COVID-19 infection, and were thus considered COVID-naïve.We stratified the study population according to previous SARS-CoV-2 exposure into patients with prior COVID-19 infection before vaccination (n = 145) . There wp < 0.001), and remained so at every monthly assay. Serological changes in patients with previous COVID-19 exposure and in COVID-naïve patients is represented in p < 0.001).The proportion of patients who achieved seroconversion was similar in both groups . Howeverp = 0.002), and these differences remained for the following 4 months after the initial two-dose vaccine. Nonetheless, after the third SARS-CoV-2 vaccine dose, the proportion of patients who had positive anti-S(RBD) antibody levels was matched in both groups  had received a full HBV vaccination regimen, of which 84 patients (68.9%) had an effective humoral response. h groups , but theh groups .p < 0.001)  antibodies of 33 ± 14.5%. This decrease was significantly steeper in patients who were COVID-naïve (36.5 ± 11% per month) compared to patients with previous exposure to COVID-19 (17.5 ± 6%) (< 0.001) .p < 0.001). Although the rate of antibody level decline remained significantly lower in patients with previous exposure to COVID-19 , COVID-naïve patients also showed a more sustained humoral response after the third dose compared to the initial vaccination, with a monthly decrease in the rate of antibody level decline from 36.5 ± 11 to 27.8 ± 16% (p < 0.001). Also, patients who were responders to HBV vaccines showed a more sustained response after the third dose, with a decrease in the rate of antibody decline from 33.5 ± 12.3 per month to 24.7 ± 16% per month (p < 0.001) (The mean monthly rate of antibody titer decline decreased to 25 ± 16.7% after the third dose (< 0.001) .We analyzed factors that were associated with an effective humoral response after the initial two-dose mRNA vaccination. Patients with previous exposure to COVID-19 , previous humoral response to HBV vaccines , patients who were < 70 years old  and patients who received mRNA-1273  were associated with an effective sustained humoral response after 4 months from vaccination in the univariate analysis. Patients with history of neoplasms were less likely to maintain an effective humoral response . In the multivariable linear regression model, both previous exposure to COVID-19 infection and previous humoral response to HBV vaccines maintained statistical significance. Details are presented in In the univariate analysis to determine the factors associated with a sustained humoral response after 4 months from receiving the third dose of mRNA vaccine, we found an association with response to HBV vaccines , and a tendency for a less likely effective seroconversion in patients receiving immunosuppressants . After adjusting for age, dialysis vintage, immunosuppression, and type of vaccine administered, the humoral response to HBV vaccines remained as an independent factor associated with an effective sustained humoral response after 1 year from the initial vaccination. All patients with prior exposure to COVID-19 developed an effective humoral response, and therefore could not be included as a factor in the multivariate analysis .Throughout the year following SARS-CoV-2 vaccination, there were 23 vaccinated patients (12.9%) who were diagnosed with SARS-CoV-2 infection, of which 3 cases (1.7%) were after receiving the first two doses, and the remaining cases (11.2%) were diagnosed after receiving the third dose of vaccination. Twenty-one patients (91.3% of the infected patients) remained asymptomatic. There were only two cases (1.1%) of severe COVID-19 with acute respiratory failure requiring hospital admission and high flow oxygen therapy among vaccinated patients included in this study, both of which had an inadequate humoral response to the vaccine. The first patient was an 85-year-old male patient who became infected 3 months after receiving the first two mRNA vaccine doses, having reached a peak SARS-CoV-2 anti-S(RBD) antibody titer of 1,007 AU/ml in the first month after vaccination, and decreasing to 510 AU/ml in the second month, just before infection. The second patient was a 72-year-old female patient under immunosuppression with tocilizumab due to severe rheumatoid arthritis. She never responded to the initial two doses of mRNA vaccine nor to the booster dose (peak SARS-CoV-2 anti-S-RBD antibodies 1 AU/ml) and was infected 3 months after receiving the third dose. Neither patient had a history of COVID-19 infection before vaccination, and both patients died.In this serological study we examined the monthly changes in SARS-CoV-2 anti-S(RBD) IgG levels for 1 year after vaccination. We observed that patients who were COVID-naïve and those who had not previously responded to HBV vaccines benefited the most from a third mRNA vaccine dose against SARS-CoV-2, since it provided them with an effective humoral response , both immediately after vaccination and also sustained in time for at least 4 months after receiving the booster dose, whereas the first two doses had been unable to elicit such a sustained response.Several studies have determined the factors that are associated with an immediate humoral response few weeks after vaccination, including serum albumin levels, lymphocytes, dialysis vintage, intravenous iron dose, immunosuppressant treatment, previous infection with COVID-19, and a positive response to HBV vaccines –22. In op = 0.009). In this way, 4 months after receiving the booster dose, there were no significant differences in the protective humoral response between COVID-naïve patients and those who were previously infected with COVID-19 .Naïve patients for COVID-19 infection are the ones who benefit the most from vaccination and especially from the administration of a third dose . In our During the study period, the accumulated incidence in our area was around 20,500 cases per 100,000 population , predomip < 0.001). Therefore, this group of patients not only benefits from the first two vaccine doses but also from the booster effect of a third dose, although to a lesser extent than in the COVID-naïve population.HD patients who have had a COVID-19 infection have less protection against reinfection than the general population . HoweverPrevious studies have compared the humoral response after the second and third doses. One study analyzed the humoral response after the third dose of the vaccine, showing a slight increase in the proportion of patients who achieve seroconversion, but with a very intense humoral response, reaching a 36-fold increase in median antibody titers after the booster dose , and anoIt has been previously described that there is an association between humoral responses after HBV and SARS-CoV-2 vaccines in HD , 32, 33.It should be noted that the most used vaccine in our patients was mRNA-1273, 77.5% of the patients for the first two doses and 91.7% for the third dose (with no significant differences between the different groups of patients). It has been described that the type of vaccine acts as an independent factor for response to vaccination and that mRNA-1273 is the one that obtains the best results, perhaps due to its higher dose of mRNA .We believe that in the future, the rate of antibodies will continue to decline and a significant percentage of patients will probably lose protection in the mid-term. Therefore, monitoring the levels of antibodies in the HD population should be a priority in order to propose new immunizations when necessary, as other authors have suggested .Consequently, we could revaccinate when SARS-CoV-2 anti-S(RBS) IgG levels drop below a certain threshold, in a similar way to how revaccination of HBV is recommended when anti-HBs antibodies drop below 10 mIU/mL. We suggest that monitoring of anti-S(RBD) IgG levels should be particularly frequent in patients who did not respond to HBV vaccines, in COVID-naïve patients and in those with lower antibody levels.Our study has a number of limitations. First, the rate of neutralizing antibodies was not determined, which according to other authors can decrease progressively after the first two doses and can rise very significantly after the third dose. However, other studies have found a significant correlation between anti-S(RBD) antibodies and the neutralizing antibody response, and in our study we found that the changes in anti-S(RBD) IgG antibody levels resemble those described in neutralizing antibodies , 19. AnoIn conclusion, as a consequence of receiving a third dose of SARS-CoV-2 mRNA vaccine, HD patients achieve a better and more sustained humoral immune response over time, which probably translates into lower morbidity and mortality rates in SARS CoV-2 infection. Patients who are most likely to benefit from the booster dose are COVID-naïve patients and patients who were non-responders to HBV vaccines.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Medical Research Ethical Committee (CEIm), Hospital Universitario Fundación Alcorcón. The patients/participants provided their written informed consent to participate in this study.EG-V and AS: conceptualization, writing—original draft preparation, and writing—review and editing. EG-V: methodology and project administration. EG-V, AS, and ML-P: validation. AS: formal analysis. JA-S: investigation. EG-V and EG: resources. EG-V and ML-P: data curation. JA-S, AC, CC-C, and GF-J: visualization. All authors have read and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.